Seed-specific promoter from the rice glutelin GluB-1 gene and uses thereof

ABSTRACT

An objective of the present invention is to provide promoters having seed-specific promoter activity, and methods of expressing foreign proteins in seeds. The present inventors isolated the promoters of a number of genes that are expressed in rice seeds, constructed binary vectors in which each promoter is inserted upstream of the GUS reporter gene, and transformed rice using the  Agrobacterium  method. In particular a seed-specific promoter from the rice glutelin GluB-1 gene is disclosed. GUS expression level was used as an index to examine the site of expression, the expression pattern during seed maturation, and the level of expression in seeds for each promoter. Promoters with activity specific to a particular site in seeds, and with higher activity than constitutive promoters and known seed-specific promoters were discovered.

RELATED APPLICATIONS

This application claims priority to JP 2003-373815, filed Oct. 31, 2003,which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to seed-specific gene promoters and uses thereof.

BACKGROUND OF THE INVENTION

Recombinant DNA technology is being implemented as a way of improvingplant breeds. Using this technology, plants with additional functionssuch as herbicide resistance, pest insect resistance, and the like havebeen created, and progress is being made in their practical application.The use of recombinant technologies to improve plant breeds not onlyaims to add new functions to plants: there has been much research anddevelopment into the expression of useful proteins in plants, byintroducing these plants with a foreign gene. Such research uses plantsas factories to produce useful proteins.

Production of recombinant proteins in plants has many advantages, themost evident of which are the reduced cost compared to systems thatutilize transgenic animals; the ease with which scale of production canbe adjusted to suit market size; and the absence of any risk ofcontamination by animal-borne pathogens such as viruses and prions(Daniell et al., Trends Plant Sci., 6, 219–226 (2001); Fischer andEmans, Transgenic Research, 9, 279–299 (2000); Giddings et al., NatureBiotech., 18, 1151–1156 (2000)).

Recently, systems using seeds for production of recombinant protein inplants have been shown to be more advantageous than those using leavesor roots (Delaney, 2002, Plants as Factories for Protein Production(Hood, E. E. and Howard, J. A) pp. 139–158 (2002). Netherlands: KluwerAcademic; Howard and Hood, Plants as Factories for Protein Production(Hood, E. E. and Howard, J. A) pp. vii–x (2002). Netherlands: KluwerAcademic). Seeds are storage organs, in which a special organelle calleda protein body stably stores a large amount of a small number of storageproteins. This feature has been employed by using seeds as idealbioreactors for producing recombinant protein. Recombinant proteinsaccumulated in seeds are very stable, and can be administered orallywithout any need for further processing or purification. Antibodies orvaccines expressed in seeds are reported to be highly stable, and can bestored for years, even at room temperature, without decomposition.Moreover, vaccines administered via seeds are thought to triggerantibody production by the mucosal immune system, without processing orpurification (Walmsley and Arntzen, Curr. Opin. Biotech., 11, 126–129(2000)).

When producing proteins using recombinant technology, the yield of aprotein of interest is affected by many factors, including transcriptionfactors. The most important and easily controlled of these factors isthe choice of promoter. In order to use rice seeds as a platform forrecombinant protein production, it is important to use a promoter suitedto the needs of individual proteins and their use in biotechnology. Thisis because the promoter controls the timing, location and level ofexpression.

However, analyses of the cis-regulatory factors involved inendosperm-specific expression are limited to those of a small number ofglutelin genes, using different species (transgenic tobacco) and thesame species (transgenic rice). (Croissant-Sych and Okita, Plant Sci.,116, 27–35 (1996); Takaiwa et al., Plant Mol. Biol., 16, 49–58 (1991a);Takaiwa et al., Plant Mol. Biol., 30, 1207–1221 (1996); Wu et al., PlantJ., 14, 673–983 (1998a); Wu et al., Plant J., 23, 415–421 (2000);Yoshihara et al., FEBS Lett., 383, 213–218 (1996); Zhao et al., PlantMol. Biol., 25, 429–436 (1994); Zheng et al., Plant J., 4, 357–366(1993)). Studies of a few other rice storage protein promoters were nomore than observations of their spatial expression patterns (Wu et al.,Plant Cell Physiol., 39, 885–889 (1998b)).

SUMMARY OF THE INVENTION

The present invention has been made considering the above circumstances.An objective of the present invention is to provide promoters withseed-specific promoter activity, and methods of expressing foreignproteins in seeds. Another objective is to provide promoters withspecific promoter activity in a particular site, such as the seedendosperm, embryo, and aleurone layer.

In order to achieve the above objectives, the present inventors isolateda number of promoters of rice genes expressed in seeds, and constructedbinary vectors in which each promoter was inserted upstream of GUSreporter gene. The present inventors then transformed rice using theAgrobacterium method. Then, for each promoter, the inventors used GUSexpression as an index to examine the site of expression, the expressionpattern during seed maturation, and the level of expression in seeds.They thus discovered promoters with an activity of expression specificto a particular site in seeds, and with higher activity thanconstitutive promoters and known seed-specific promoters. As describedabove, it is useful for a seed expressing a foreign gene product to betaken as food. However, for this to be possible, the foreign gene mustbe expressed in an edible part of the seed. For example, in one of themain cereals, rice, the endosperm is normally eaten, and therefore theabove goal would be achieved by using an endosperm-specific promoter inrice. Furthermore, promoters specific for a particular site in a seedwill enable expression of a foreign gene at a desired place in a seed.Therefore, these promoters can be valuable tools for metabolicengineering using seeds. For example, using a promoter that directsexpression in the aleurone layer or embryo may control fatty acidmetabolism.

Thus, the present invention relates to promoters specific to aparticular site in a seed, and to uses thereof. More specifically, itprovides:

-   -   (1) a DNA comprising promoter activity in seeds, wherein the DNA        is any one of the following (a) to (c):    -   (a) a DNA comprising a nucleotide sequence of any one of SEQ ID        NOs: 1 to 7,    -   (b) a DNA comprising a nucleotide sequence wherein one or more        nucleotides are added, deleted, substituted, or inserted into a        nucleotide sequence of any one of SEQ ID NOs: 1 to 7, and    -   (c) a DNA that hybridizes under stringent conditions with a DNA        comprising a nucleotide sequence of any one of SEQ ID NOs: 1 to        7;    -   (2) a DNA comprising a gene functionally linked downstream of        the DNA of (1);    -   (3) a vector comprising the DNA of (1) or (2);    -   (4) a transformed plant cell carrying the DNA of (2);    -   (5) a transformed plant cell introduced with the vector of (3);    -   (6) a transformed plant carrying the cell of (4) or (5);    -   (7) a reproductive material of the plant of (6);    -   (8) the reproductive material of (7), wherein the reproductive        material is a seed; and    -   (9) a method of expressing a gene in a seed generated from a        plant cell, comprising the steps of:        -   (a) introducing the DNA of (2) or the vector of (3) into the            plant cell, and        -   (b) regenerating a plant from the plant cell.

In certain preferred embodiments, the isolated DNA described abovefurther includes a 3′-untranslated region as shown in SEQ ID NO: 8.

Certain embodiments of the invention are directed to an isolated DNAwhich confers seed-specific gene expression, where the DNA is any one ofthe following

-   -   (a) to (c):    -   (a) a DNA which includes a nucleotide sequence of SEQ ID NO: 8,    -   (b) a DNA which includes a nucleotide sequence where one or more        nucleotides are added, deleted, substituted, or inserted into a        nucleotide sequence of SEQ ID NO: 8, and    -   (c) a DNA that hybridizes under stringent conditions with a DNA        which includes a nucleotide sequence of SEQ ID NO: 8. Also        included in certain embodiments of the invention is a vector        which includes an isolated DNA as described above.

A preferred embodiment of the invention is directed to a vector whichincludes the promoter of SEQ ID NO: 3 and the 3′-untranslated region ofSEQ ID NO: 8.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of the construction of the chimeric gene used forrice transformation. The 5′-flanking regions of various genes encodingrice seed storage proteins and non-storage proteins were fused into aregion between two restriction sites, selected from HindIII, SalI, andSmaI sites. The GUS reporter gene and the Nos terminator were thenfused. The promoter was shown to promote the genes: 1.3 kb GluB-1, 2.3kb GluB-1, GluB-2, GluB-4, 10 kDa prolamin, 13 kDa prolamin (PG5), 16kDa prolamin, 26 kDa Glb-1, REG2, Ole18, soybean β-conglycinin, AlaAT,GOGAT, PPDK, AGPase, and SBE1.

FIG. 2 is a series of pictures depicting the results of histochemicalanalysis of GUS expression induced by the gene promoters of various seedstorage proteins and non-storage proteins. The GUS protein was detectedby vertically dissecting transgenic seeds by hand, and incubating thesections in a solution containing X-Gluc. a, 1.3 kb GluB-1 promoter; b,2.3 kb GluB-1 promoter; c, GluB-2 promoter; d, GluB-4 promoter; e, 10kDa prolamin promoter; f, 13 kDa prolamin (PG5a) promoter; g, 16 kDaprolamin promoter; h, 26 kDa Glb-1 promoter; i, REG2 promoter; j, Ole18promoter; k, β-conglycinin promoter; 1, AlaAT promoter; m, GOGATpromoter; n, AGPase promoter; o, PPDK promoter; and p, SBE1 promoter.

FIG. 3 is a series of pictures showing the results of histochemicalanalysis of GUS expression in vegetative tissues. If, leaf, Is, leafsheath; sk, stalk; rt, root; and ed, eudodersis. a, 10 kDa prolaminpromoter; b, PPDK promoter; and c, AGPase promoter.

FIG. 4 is a series of pictures showing the time course of changes in GUSactivity induced by seed promoters, over the maturation stages of seeddevelopment. It shows the results of histochemical staining, usingX-Gluc, of the vertical sections of transgenic rice seed at 7, 12, and17 DAF. a, 1.3 kb GluB-1 promoter; b, 2.3 kb GluB-1 promoter; c, GluB-2promoter; d, GluB-4 promoter; e, 10 kDa prolamin promoter; f, 13 kDaprolamin (PG5a) promoter; g, 16 kDa prolamin promoter; h, 26 kDa Glb-1promoter; i, REG2 promoter; and j, Ole18 promoter.

FIG. 5 continues from FIG. 4 and is a series of pictures. k,β-conglycinin promoter; 1, AlaAT promoter; m, GOGAT promoter; n, AGPasepromoter; o, PPDK promoter; p, SBE1 promoter; and q, ubiquitin promoter.

FIG. 6 shows the results of measuring the GUS activity expressed by thevarious promoters in maturating seed at 17 DAF. GUS activity isexpressed in pmol 4MU/min/μg protein units.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel DNAs with promoter activity inseeds. This invention, as described above, is based on the discovery bythe present inventors of promoters with promoter activity specific toparticular sites in seeds, and that exhibit greater activity thanconstitutive promoters and known seed-specific promoters.

Specifically, the above DNAs of the present invention include DNAs withpromoter activity that comprise a sequence of any of SEQ ID NOs: 1 to 7.The present inventors identified these DNAs as novel rice-derived DNAscomprising promoter activity, and grouped them into the following threegroups:

-   -   Promoter DNAs specific to the endosperm (the nucleotide        sequences of the respective DNAs are shown in SEQ ID NOs: 1 to        4).    -   Promoter DNAs specific to embryo or aleurone tissue (the        nucleotide sequences of the respective DNAs are shown in SEQ ID        NOs: 5 and 6).    -   Promoter DNAs for expression in the entire seed (the nucleotide        sequence of the DNA is shown in SEQ ID NO: 7).

Group (A), the endosperm-specific promoter DNA group, comprises the riceglutelin GluB-1 gene promoter (SEQ ID NO: 1), rice glutelin GluB-4 genepromoter (SEQ ID NO: 2), 10 kDa prolamin promoter (SEQ ID NO: 3), and 16kDa prolamin promoter (SEQ ID NO: 4). Expression of this group can beobserved in aleurone and sub-aleurone tissues at seven days afterflowering, and progressively spreads into the inner endosperm regionduring maturation. This expression pattern does not change during thematuration process.

Group (B), the embryo or aleurone tissue-specific promoter DNA group,comprises the rice embryo globulin gene promoter (SEQ ID NO: 5), andrice oleosin promoter (SEQ ID NO: 6). This group shows expression in thealeurone tissue in the early stages of maturation (seven days afterflowering), and the expression spreads into the embryo and aleuronetissue during maturation, but not to the endosperm.

Group (C), promoter DNAs for expression in the entire seed, comprisesthe rice ADP-glucose pyrophosphorylase gene promoter (SEQ ID NO: 7).This promoter first shows expression in the embryo in the early stage ofmaturation, and then in the entire seed during maturation (expression inthe embryo is also extremely high in the late stage of maturation).

One skilled in the art can use conventional methods to prepare DNAscomprising the seed-specific promoters of groups (A) to (C) (hereinafterabbreviated as “the DNAs of this invention”). For example, the DNAs canbe prepared by designing an appropriate pair of primers based on anucleotide sequence of any of SEQ ID NOs: 1 to 7 (for example, SEQ IDNOs: 9 to 22), and performing PCR using a rice genomic DNA as thetemplate, and screening a genomic library with the resulting amplifiedDNA fragment as a probe. Moreover, a commercially available DNAsynthesizer may be used to synthesize a desired DNA.

The DNAs of this invention may be used to obtain (isolate) DNAscomprising promoter activity. In the first step of isolating a DNA, aDNA of this invention or its part may be used as a probe, or anoligonucleotide that specifically hybridizes with a DNA of the inventionmay be used as a primer to isolate a DNA comprising high homology withthe above DNA from a desired organism. The DNAs of this invention alsocomprise DNAs that hybridize with DNAs comprising a nucleotide sequenceof any of SEQ ID NOs: 1 to 7, which can be isolated using standardhybridization techniques (Southern E. M., J. Mol. Biol., 98, 503 (1975))or PCR methods (Saiki R. K. et al., Science, 230, 1350 (1985); Saiki R.K. et al., Science, 239, 487 (1988)). Thus, it is feasible for oneskilled in the art to isolate from a desired organism a DNA highhomologous to a DNA comprising a nucleotide sequence of any of SEQ IDNOs: 1 to 7, using a DNA comprising a nucleotide sequence of any of SEQID NOs: 1 to 7 or its part as a probe, or an oligonucleotide thatspecifically hybridizes with a DNA comprising a nucleotide sequence ofany of SEQ ID NOs: 1 to 7 as a primer. In order to isolate such DNAs,hybridization is preferably performed under stringent conditions.Hybridization may be performed with buffers that permit the formation ofa hybridization complex between nucleic acid sequences that contain somemismatches. At high stringency, hybridization complexes will remainstable only where the nucleic acid molecules are almost completelycomplementary. Many factors determine the stringency of hybridization,including G+C content of the cDNA, salt concentration, and temperature.For example, stringency may be increased by reducing the concentrationof salt or by raising the hybridization temperature. Temperatureconditions for hybridization and washing greatly influence stringencyand can be adjusted using melting temperature (Tm). Tm varies with theratio of constitutive nucleotides in the hybridizing base pairs, andwith the composition of the hybridization solution (concentrations ofsalts, formamide and sodium dodecyl sulfate). In solutions used for somemembrane based hybridizations, addition of an organic solvent, such asformamide, allows the reaction to occur at a lower temperature.Accordingly, on considering the relevant parameters, one skilled in theart can select appropriate conditions to achieve a suitable stringencybased experience or experimentation. Herein, stringent hybridizationconditions mean conditions using 6 M urea, 0.4% SDS, and 0.5×SSC, orthose using 0.1% SDS (60° C., 0.3 M NaCl, 0.03 M sodium citrate), orconditions providing an equivalent stringency. Under more stringentconditions, for example, performing hybridization in 6 M urea, 0.4% SDS,and 0.1×SSC, one can expect to isolate DNAs with higher homology. Highhomology means sequence identity over the entire nucleotide sequence ofpreferably 50% or higher, more preferably, the isolated DNA is at leastabout 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more, identical.

To determine the percent identity of two DNAs, the sequences are alignedfor optimal comparison purposes. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences. The percent identity between two sequences can bedetermined using conventional techniques such as to those describedherein, with or without allowing gaps. In calculating percent identity,typically exact matches are counted. For example, when an isolated DNAof the present invention is longer than or equivalent in length to aprior art sequence, the comparison is made with the full length of theinventive sequence. Alternatively, when an isolated DNA of the presentinvention is shorter than the prior art sequence, the comparison is madeto a segment of the prior art sequence of the same length as that of theinventive sequence (excluding any loop required by the homologycalculation).

Identity between nucleotide sequences can be determined by using theBLAST algorithm developed by Karlin and Altschul (Proc. Natl. Acad. Sci.U.S.A., 87, 2264–2268 (1990); Karlin S. and Altschul S. F., Proc. Natl.Acad. Sci. U.S.A., 90, 5873). A program called BLASTN has been developedbased on the BLAST algorithm (Altschul S. F. et al., J. Mol. Biol., 215,403 (1990)). When analyzing nucleotide sequence using BLASTN, parametersmay be set as score =100 and wordlength=12, for example. When using theBLAST and Gapped BLAST programs, the default parameters for each programmay be chosen. Specific procedures for these analyses are publiclyknown. Another example of a mathematical algorithm that may be utilizedfor the comparison of sequences is the algorithm of Myers and Miller(1988) CABIOS 4:11–17.

The DNAs of this invention are normally derived from plants, preferablyfrom monocotyledons, and more preferably from Poaceae, but are notlimited to any particular origin, as long as the DNA has seed-specificpromoter activity.

In addition, this invention provides DNAs that are structurally similarto the above DNAs, and comprise promoter activity. Such DNAs includeDNAs with seed specific promoter activity that comprise a nucleotidesequence wherein one or more nucleotides are substituted, deleted,added, and/or inserted into a nucleotide sequence of any of SEQ ID NOs:1 to 7. Such DNAs can also be used to isolate a DNA of this inventionthat comprises promoter activity. Methods for preparing such DNAs arewell known to those skilled in the art, and include the hybridizationtechniques and polymerase chain reaction (PCR), as described above.Furthermore, the above DNAs may be prepared by introducing mutationsinto DNAs comprising a nucleotide sequence of any of SEQ ID NOs: 1 to 7,for example, by using site-directed mutagenesis method (Kramer W. andFritz H. J., Methods Enzymol., 154, 350 (1987)).

One skilled in the art can determine whether or not the DNAs prepared asabove comprise promoter activity by using methods such as known reporterassays using reporter genes. Reporter genes are not limited to anyparticular gene, as long as their expression is detectable. For example,reporter genes may be those routinely used by those skilled in the art,such as CAT gene, lacZ gene, luciferase gene, β-glucuronidase (GUS)gene, and GFP gene.

The reporter gene expression level can be measured by the methodscommonly known to those skilled in the art, depending on the type ofreporter gene. For example, when CAT gene is used as the reporter gene,the expression level of the reporter gene can be measured by detectingacetylation of chloramphenicol by the gene product. When lacZ gene isused as the reporter, expression level can be measured by detecting thecolor of a dye compound produced by the catalytic function of the geneproduct. In the case of luciferase gene, expression level can bemeasured by detecting fluorescence from a fluorescent compound producedby the catalytic function of the gene product; the GUS reporter geneexpression can be measured by detecting luminescence of Glucuron (ICN),or the color of 5-bromo-4-chloro-3-indolyl-β-glucuronide (X-Gluc) as theresult of catalytic function of the gene product. Furthermore, GFPexpression can be measured by detecting the fluorescence of GFP protein.

In addition, if a gene other than those described above is used as areporter, the expression level of the gene can be measured by methodsknown to those skilled in the art. For example, mRNAs may be extractedby standard methods, and then used as templates to perform Northernhybridization or RT-PCR to measure the transcription level of the gene.Furthermore, DNA array technology may be used to measure genetranscription levels. In addition, fractions containing proteins encodedby the genes may be recovered by standard methods, and expression of theprotein of the present invention may be detected by electrophoresis,such as SDS-PAGE, to measure the translation level of the gene.Furthermore, the expression of the protein encoded by a gene may bedetected by Western blotting, using an antibody against the protein tomeasure its translation level. The antibody used to detect the proteinencoded by the gene can be any antibody, and is not particularly limitedas long as it is detectable. For example, both monoclonal antibodies orpolyclonal antibodies may be used. The antibody can be prepared bymethods known to those skilled in the art.

Furthermore, the present invention provides DNAs in which an arbitrarygene is functionally linked downstream of an above promoter DNA. TheDNAs of the invention enable specific expression of a desired protein orpeptide, encoded by an arbitrary gene, in seeds, by activating apromoter DNA.

Herein, “functionally linked” means that a DNA of the invention and agene are linked to each other such that expression of the downstreamgene is triggered by binding of a transcription factor to a DNA withpromoter activity of the present invention. Thus, even if the gene islinked with another gene, and forms a fusion protein with the product ofthe other gene, it is collectively considered to be “functionallylinked” as long as expression of the fusion protein is induced when atranscription factor binds to the DNA of this invention.

The present invention also provides vectors comprising a DNA (referredto below as “the above DNA”), wherein an arbitrary gene is functionallylinked to an above promoter DNA or downstream of it. The vectors of thisinvention are useful for maintaining the above DNA in host cells, or forexpressing a protein of interest and such by transforming plants.

The vectors used for insertion of the above DNA are not limited to anyparticular vector as long as they enable expression of the inserted genein plant cells. For example, vectors comprising a promoter forconstitutive gene expression in plant cells (for example, cauliflowermosaic virus 35S promoter), or vectors comprising a promoter that can beactivated by an external stimuli in an inducible manner, can be used.Vectors comprising the above DNA as an insert may be introduced intoplant cells by methods commonly known to those skilled in the art, suchas polyethylene glycol methods, electroporation methods,Agrobacterium-mediated methods, and particle gun methods.Agrobacterium-mediated methods may be performed, for example, by themethod of Nagel et al. (Microbiol. Lett., 67, 325 (1990)), byintroducing an expression vector comprising the above DNA as an insertinto Agrobacterium, and infecting plant cells with the Agrobacterium bydirect infection or the leaf disc method, to introduce the above DNAinto plant cells.

In addition, the present invention provides transformed plant cells intowhich the above DNA or vector is introduced. The transformed plant cellsof the present invention can be any form of plant cell, or a cluster ofplant cells into which the above DNA or vector is introduced, as long asthe cells can regenerate a plant. For example, the plant cells of thepresent invention comprise suspensions of cultured cells, protoplasts,leaf sections, and calluses.

Vectors can be introduced into plant cells by a variety of methodscommonly known to those skilled in the art, such as by polyethyleneglycol methods, electroporation methods, Agrobacterium-mediated methods,and particle gun methods.

Furthermore, the present invention provides transformed plants carryingthe above-described cells. The plants of the invention can be used insystems for producing a desired gene product.

Plants can be regenerated from transformed plant cells using methodsknown commonly to those skilled in the art, according to the type ofplant. For example, rice may be regenerated by the method of Fujimura etal. (Plant Tissue Culture Lett., 2, 74 (1995)), corn may be regeneratedby the method of Shillito et al. (Bio/Technology, 7, 581 (1989)) or themethod of Gorden-Kamm et al. (Plant Cell, 2, 603 (1990)), and potato maybe regenerated by the method of Visser et al. (Theor. Appl. Genet., 78,594 (1989)). Arabidopsis may be regenerated by the method of Akama etal. (Plant Cell Reports, 12, 7–11 (1992)), and eucalyptus may beregenerated by the method of Doi et al. (JP-A Hei 8-89113).

Furthermore, the present invention provides not only plants carryingcells into which the above DNA has been introduced, but also thereproductive materials of those plants. After obtaining transformedplants that have the above DNA or vector introduced in their genome,reproductive materials (for example, seeds, fruits, cuttings, tubers,tuberous roots, shoots, calluses, and protoplasts) can be obtained, andthe plants can be mass-produced from these sources. In particular, inaddition to being a reproductive material, seeds are places where theabove introduced promoter causes accumulation of foreign gene product.

Furthermore, the present invention provides methods of expressing anarbitrary gene in plant seed cells. The methods of the inventioncomprise the steps of introducing plant cells with a DNA in which anarbitrary gene is functionally linked downstream of the above promoterDNA of the invention, or the above vector of this invention, andregenerating a plant from the plant cells. The steps of introductioninto plant cells and regeneration of a plant can be performed by theabove methods. The methods of the invention may be used to obtain adesired gene product from a plant, or to obtain seeds that accumulatethe desired gene product. Plants regenerated using a desired gene by amethod of this invention bear seeds that accumulate the product of thedesired gene. Thus, the gene product can be obtained by purificationfrom these seeds, or such. In addition, if the desired gene product is amedicinal compound or the like, seeds can be used as a final form,omitting purification steps, because seeds can be ingested as is.

As used herein, an “isolated promoter” is a promoter removed from itsoriginal environment (e.g., the natural environment if naturallyoccurring) and thus, altered by the “hand of man” from its naturalstate.

Of the promoters isolated by the present inventors, 10 kDa rice prolaminpromoter resulted in an interesting observation. In testing seedspecific promoter activity, it was found that when the Nos terminatorwas linked downstream of 10 kDa prolamin promoter, gene expression wasinduced not only in seeds but also in the phloem of roots, stalks, andthe like. However, when the original 3′-untranslated region (0.3 kb; SEQID NO: 8) was linked, gene expression other than in seeds was clearlysuppressed. Thus, the inventors discovered that the 3′-untranslatedregion of the rice 10 kDa prolamin promoter can suppress gene expressionin tissues other than seed endosperm, and is therefore required forendosperm-specific gene expression. The inventors had previously foundthat a foreign gene product could be expressed in seeds at a high levelby inserting the 5′-untranslated region between a promoter ensuringexpression in seeds and a foreign gene (JP-A 2002-58492). This seedstorage protein gene is the first case for which both the 3′-flankingregion and the 5′-flanking region have been identified as necessary forseed specific expression.

Endosperm-specific gene expression, where gene expression in tissuesother than endosperm is suppressed, is made possible by inserting the3′-untranslated region downstream of a promoter that comprises activityin tissues in addition to endosperm.

Thus, the present invention further provides (1) DNAs comprising a3′-untranslated region of SEQ ID NO: 8, (2) vectors comprising the3′-untranslated region, (3) vectors comprising a promoter and the3′-untranslated region, (4) vectors comprising a promoter, a gene, andthe 3′-untranslated region, (5) cells and transformed plants carryingthe vector of (4), (6) reproductive materials of the transformed plants,and (7) methods of inducing endosperm-specific gene expression by usingthe 3′-untranslated region.

Seeds are essentially storage organs, and contain a large space foraccumulating foreign gene products. Enzymes, antibodies, and the likethat are accumulated in seeds are stable for more than one year, evenwhen stored at room temperature. In addition, there is no need to purifysuch proteins if they are taken as food. Thus, useful products can beproduced for extremely low costs. Furthermore, no special productionfacilities are required, other than agricultural fields, and they aresafe from the risk of contamination by animal viruses and such.

The promoters of the present invention are especially valuable aspromoters for the production of useful products using seeds. Forexample, these promoters enable the large-scale production of medicinalproducts (e.g., vaccines, antibodies, blood products, and interferons)or industrial enzymes in seeds. In addition, they can be used to expressallergen epitopes in plants, creating plant crops for the treatment ofallergies such that eating the seed of such a plant can treatpollinosis, house dust allergies, and the like. These promoters alsoenable the expression in a seed of a foreign gene whose product ishighly nutritious, thus improving the nutritional value of the seed.Furthermore, it is possible to use the above promoters to createfunctional seeds by expressing functional peptides or functionalproteins that comprise the effect of reducing high blood pressure, serumcholesterol, blood sugar, or such in seeds.

In addition, the set of promoters of this invention, which enable theexpression of a gene in a desired region of a seed and a desired stageof seed development, can be used as important tools for metabolicengineering utilizing seeds. For example, by using a promoter thatdirects expression to the aleurone layer or embryo, a metabolic processof interest in fatty acid metabolism can be controlled.

Any patents, patent applications, and publications cited herein areincorporated by reference.

EXAMPLES

This invention will be explained in detail below with reference toExamples, but it is not to be construed as being limited thereto.

Example 1 Construction of the Promoter-GUS Gene Chimeric Constructs andIsolation of Transgenic Plants

The expression patterns and promoter activity of a number of genesexpressed in seeds were characterized, instead of examining the genespresumed to be regulatory factors. Fifteen different promoters rangingin size from 0.8 to 2.4 kb were isolated by PCR using genomic DNA orgenomic clones as a template.

The genes and the size of their corresponding promoters are as follows:rice 10 kDa prolamin, 0.8 kb; rice 13 kDa prolamin (PG5a), 0.9 kb; rice16 kDa prolamin, 0.9 kb; rice glutelin GluB-4, 1.4 kb; rice embryoglobulin (REG2), 1.3 kb; rice 18 kDa oleosin (Ole18), 1.3 kb; riceglutamate synthase gene (GOGAT), 0.8 kb; rice pyruvate orthophosphatedikinase (PPDK), 0.8 kb; rice ADP-glucose pyrophosphorylase (AGPase),2.0 kb; rice starch branching enzyme (SBE1), 2.0 kb; and soybeanβ-conglycinin, 1.0 kb. Rice glutelin GluB-1, 1.3 kb, 2.3 kb; riceglutelin GluB-2, 2.4 kb; rice alanine aminotransferase (AlaAT), 1 kb;rice 26 kDa globulin (Glb-1), 1.0 kb; and maize ubiquitin promoter, 2kb.

Of these, the promoter sequences of 2.3 kb GluB-1, GluB-4, 10 kDaprolamin, 16 kDa prolamin, rice embryo globulin, rice oleosin, and riceADP-glucose pyrophosphorylase are shown by SEQ ID NOs: 1 to 7, and thesequences of the primer pairs used to isolate these promoters are shownby SEQ ID NOs: 9 to 22, respectively.

Fragments of various promoters were inserted into the modified binaryvector pGPTV-35S-HPT, which comprises the hygromycin phosphotransferase(HPT) gene as a selection marker (FIG. 1). The modified vector wasconstructed from the pGPTV-HPT binary vector (Becker et al. (I 992))using the Nos promoter as the HPT gene promoter instead of the 0.8 kbCaMV35S promoter. The seed gene promoters to be tested were introducedupstream of the UdiA gene encoding β-glucuronidase (GUS) in the modifiedbinary vector.

Transgenic rice plants (Oryza sativa cv Kitaake) were created usingAgrobacterium-mediated transformation. The plasmids constructed as abovewere introduced into EHA 105 strain Agrobacterium tumefaciens byelectroporation. Five-week-old calluses derived from mature rice seedswere treated with the transformed A. tumefaciens for three days. Each ofthe infected calluses was continuously cultured for four weeks in N6selection media comprising hygromycin, and MS regeneration media.Regenerated young plants were transferred to an incubator (Goto et al.,Nature Biotech., 17, 282–286 (1999)).

More than 20 different lines of independent transgenic plants weregenerated for each construct. The presence of the promoter fusions ofinterest was confirmed by PCR using genomic DNAs isolated from theleaves of independent transgenic rice lines, and the positive lines wereused to characterize the promoter.

Example 2 Activity of the Seed Storage Protein Gene Promoters in Seeds

Transgenic rice seeds were examined by histochemical staining toidentify the site of GUS reporter gene expression, which was induced bythe seed storage protein promoters. For histochemical analysis, maturingseeds in a stage 17 days after flowering (DAF) were sectioned alongtheir longitudinal axis with a razor blade, and the sections wereincubated in 50 mM sodium phosphate buffer (pH 7.0) containing 0.5 mMX-Gluc (5-bromo-4-chloro-3-indolyl-glucuronide) and 20% methanol at 37°C. The optimal incubation time for the staining reaction varied from 30minutes to overnight, depending on the level of GUS activity.

FIG. 2 shows the detected expression patterns. Rice glutelin promoters(1.3 kb and 2.3 kb GluB-1, GluB-2, and GluB-4; FIG. 2 a to d) andprolamin promoters (10 kDa, 13 kDa, and 16 kDa; FIG. 2 e to g) inducedGUS gene expression in endosperm. GUS gene expression by the glutelinpromoters and prolamin promoters was also detected in aleurone layer andsubaleurone tissues, but not in embryos. Further detailed examination ofthe maturing seeds of transgenic rice carrying the glutelin promotersand prolamin promoters revealed that the peripheral endosperm regionsshowed the highest GUS activity, while the inner regions showed weakactivity. The GluB-1 promoters (both 1.3 kb and 2.3 kb) showedsignificantly higher activity in endosperm regions close to the embryo.GUS expression induced by 13 kDa prolamin promoter (PG5a) was strictlyrestricted to the peripheral endosperm regions. The 26 kDa globulinGlb-1 promoter induced GUS expression in the inner starchy endospermtissue (FIG. 2 h). GUS expression induced by the embryo storage proteinpromoters (REG2, Ole18, and β-conglycinin; FIG. 2 i to k) was restrictedto the embryo and aleurone tissues, and was not observed in endosperm atall. The patterns of GUS gene expression induced by these embryo storageprotein promoters were almost identical. Interestingly, despite a numberof reports on differential expression between monocotyledonous anddicotyledonous plants (Chowdhury et al., Plant Cell Rep., 16, 277–281(1997); Rathaous et al., Plant Mol. Biol., 23, 613–618 (1993)), theβ-conglycinin promoter from soybean, which is a dicotyledonous plant,maintained embryo-specific expression in rice, a monocotyledon. Notably,GUS expression induced by the β-conglycinin promoter was extremely lowin rice, in sharp contrast to the high expression by the same promoterin the embryos and cotyledons of the dicotyledonous plant, tobacco.

Overall, GUS activity was not detected in any leaves, leaf sheaths,stalks, or roots of transgenic rice comprising a fusion with a seedstorage protein promoter (data not shown). The only exception was the 10kDa prolamin promoter, which induced some expression in vegetativeorgans (FIG. 3). These results support the conclusion that endospermstorage protein genes (except the 10 kDa prolamin) are expressed in anendosperm-specific manner, and the expression of embryo storage proteingenes are restricted to the embryo and aleurone layer.

Although the seed storage protein promoters resulted in specific geneexpression in the endosperm or embryo, the promoters of non-storageproteins exhibited different expression patterns (FIG. 2). The GUS genecontrolled by the AlaAT promoter was expressed in the center of starchyendosperm, and its activity was higher in the endosperm region close tothe embryo (FIG. 2 l). The expression pattern of the PPDK-GUS transgenewas similar to that of the endosperm storage proteins (FIG. 2 o). TheGUS gene controlled by the AGPase promoter was expressed over the entireseed, including the pericarp, and was highly expressed in the innerstarchy endosperm and embryo in particular (FIG. 2 n). In contrast, theGOGAT and SBE promoters induced GUS gene expression mainly in thescutellum (the of embryo and endosperm boundary) (FIGS. 2-m and 2-p).

Example 3 GUS Expression Pattern in the Vegetative Organs

Most of the examined promoters showed either endosperm- orembryo-specific GUS gene expression. However, GUS activity was alsodetected in the vegetative tissues of transgenic rice comprising thepromoters of the 10 kDa prolamin, PPDK, and AGPase genes (FIG. 3), andthose comprising the AlaAT promoter (Kikuchi et al., Plant Mol. Biol.,39, 149–159 (1999)). In these transgenic rice plants, GUS activity wasdetected in leaves, leaf sheaths, and the phloem of vascular bundles instalks, in addition to in the endosperm or over the entire seed (FIG. 3a to c). GUS activity was also detected in the endodermis of the rootsof the transgenic rice. However, the expression pattern obtained withthe AGPase promoter was slightly different from those obtained with thePPDK and 10 kDa prolamin promoters. In particular, the AGPase promoterinduced high level GUS expression in the apical meristem, whereas thelatter two induced ubiquitous staining in the root. Furthermore, theAGPase promoter showed distinct GUS activity in the root, and it wasstronger than PPDK and 10 kDa prolamin promoters.

In its natural state, the 10 kDa prolamin gene is normally expressed inendosperm undergoing maturation, and not detectable in vegetativetissues. The ectopic expression of the GUS fusion product observedherein was reversed to a normal endosperm-specific expression pattern bysubstituting the Nos terminator with the 0.3 kb region locateddownstream of the stop codon of the 10 kDa prolamin gene in its naturalstate (data not shown). Notably, this substitution of the3′-transcription termination region had almost no influence on theactivity of the promoter. These results indicate that the endospermspecific expression of 10 kDa prolamin gene requires both 5′- and3′-flanking regions.

SEQ ID NOs: 23 and 24 show the primer pair used to isolate the3′-transcription termination region.

Example 4 Promoter Activity During Seed Development

The expression pattern of introduced genes in developing seeds wasexamined by the histochemical staining of vertical sections of seedscollected at 7, 12, and 17 DAF. Specifically, seeds undergoingmaturation in stages 7, 12, and 17 days after flowering (DAF) weresectioned along their longitudinal axis with a razor blade, and the cutsections were incubated in 50 mM sodium phosphate buffer (pH 7.0)containing 0.5 mM X-Gluc (5-bromo-4-chloro-3-indolyl-glucuronide) and20% methanol at 37° C. The optimal incubation time for the stainingreaction varied from 30 minutes to overnight, depending on the level ofGUS activity.

The expression pattern during seed maturation was examined for eachtransgenic line, and FIGS. 4 and 5 show the results of eachrepresentative line for each seed promoter. Interestingly, the sitewhere GUS expression was first detected differed for each construct. Theglutelin promoter and prolamin promoter first showed blue GUS stainingin the peripheral endosperm regions, i.e., the aleurone and subaleuronetissues. In the glutelin promoter and 16 kDa prolamin promoter, stainingthen spread to the inner starchy endosperm as the seed matured (17 DAF),while this was not observed for the 10 kDa and 13 kDa prolamin promoters(FIG. 4 a to g). This expression pattern was in marked contrast to thepattern with the 26 kDa Glb-1 promoter, where blue GUS staining wasfirst detected in the inner starchy endosperm cells close to the embryo,and did not change during the development process (FIG. 4 h).

GUS gene expression induced by the REG2, Ole18, and β-conglycinin genepromoters was detected by seven days after flowering (DAF). Theiractivity tended to be observed first in the aleurone layer, and later inthe embryo. Expression by these promoters was restricted to the aleuronetissue and embryo (FIG. 4 i, j, and FIG. 5 k).

FIGS. 5 l to p show the temporal expression patterns of non-storageprotein promoters during seed maturation in representative transgeniclines. GUS expression by the AlaAT promoter was first observed in theinner starchy endosperm tissue, and eventually spread through the entireendosperm, although the embryo remained unstained (FIG. 5 l). GUSactivity by the SBE1 promoter was also restricted to the inner starchyendosperm tissue, and in particular, the tissue close to the embryo(FIG. 5 p). However, because of the extremely low level of GUS activity,the blue staining was not detectable until 12 DAF. In contrast, when theAGPase gene promoter fusion was introduced, GUS staining first appearedin the embryo, and later spread into the center of the endosperm. BlueGUS staining was finally observed all throughout seeds undergoingmaturation, with the most intense staining found in the embryo (FIG. 5n). This expression profile during seed development was very similar tothat observed for the ubiquitin promoter (FIG. 5 q). In contrast, theexpression pattern with the PPDK promoter was similar to those with theglutelin promoter and prolamin promoter (FIG. 5 o). GUS activity by theGOGAT promoter was restricted to the scutellum, and there was noparticular change during seed development, except that GUS activity wasnot detectable at 7 DAF (FIG. 5 m).

Example 5 Quantitative Analysis of the Promoter Activity

To evaluate the activity of various promoters, GUS fluorescence wasassayed by the method of Jefferson (1987). Maturing seeds at 17 DAF werehomogenized in GUS extraction buffer (50 mM NaPO4 (pH 7.01, 10 mM2-mercaptoethanol, 10 mM Na₂-EDTA, 0.1% SDS, 0.1% Triton X-100). Aftercentrifugation, 10 μl of the supernatant was mixed with 90 μl assaybuffer containing 1 mM 4-methylumbelliferyl-β-D-glucuronide (MUG). Afterincubating for one hour at 37° C., 900 μl of 0.2 M Na₂CO₃ was added tothe mixture to terminate the reaction. Values obtained using afluorometer were compared with those obtained from serial dilutions of4-methylumbelliferone (4MU). The protein amount was determined using aBIORAD Protein Assay kit, with serum albumin as the standard. Threeseeds were assayed for each transgenic plant.

As shown in FIG. 6, significant differences were found between thepromoter activities. The tested seed promoters were classified into fourgroups based on their activity. The group showing high GUS activitycomprises the following four promoters: GluB-4, 10 kDa prolamin, 16 kDaprolamin, and Glb-1 promoters. The average GUS activities of thesepromoters were 44.8±16.5, 38.8±10.8, 27.1±12.7, and 28.6±11.8 pmol4MU/min/μg protein, respectively. The group with moderate GUS activityincludes the following 2.3 kb GluB-1 and AGPase gene promoters. TheirGUS activity is lower than that observed for the high activity group,but is much higher than for the other groups. The average GUS activitiesof 2.3 kb GluB-1 and AGPase gene promoters were 21.3±7.0 and 10±4.7 pmol4MU/min/μg protein, respectively. Seven promoters, i.e., 1.3 kb GluB-1,GluB-2, 13 kDa prolamin, REG-2, Ole18, AlaAT, and PPDK promoters, weretentatively grouped into a group with relatively low GUS activity. Theaverage GUS activities of these promoters were 2.1±1.2, 5.5±2.2,7.4±5.5, 2.4±1.2, 2±4.6, 5.9±4.0, and 4.0±3.0 pmol 4MU/min/μg protein,respectively. The remaining three promoters, the GOGAT, SBE1, andβ-conglycinin gene promoters, were grouped into the low GUS activitygroup. The GUS expression induced by these promoters was very faint,with activity below 1 pmol 4MU/min/μg protein. The GUS activity of thecontrol ubiquitin promoter was an average of 7.4±8.5 pmol 4MU/min/μgprotein (in maturing seeds). Although the ubiquitin promoter has beenused in many applications as a general promoter, its level was about thesame as those obtained with the promoters of the group with relativelylow GUS activity.

For purposes of comparison, the activities of the PPDK promoter andAGPase promoter in vegetative tissues were also examined. The averageGUS activities for PPDK promoter in leaf, stalk, and leaf sheath were8.7±6.8, 3.7±3.6, and 16.3±13.9 pmol 4MU/min/μg protein respectively,and 12.5±5.0, 40.2±28.5, and 23.2±16.6 pmol 4MU/min/μg protein forAGPase promoter, respectively. The level of these promoter activitieswas about the same or even higher than those obtained with maturingseeds. In contrast, while 10 kDa prolamin promoter showed expression invegetative tissues, its GUS activity (3.1±1.1, 6.0±2.9, and 2.3±1.0 pmol4MU/min/>g protein in leaf, stalk, and leaf sheath, respectively) wassignificantly lower than that observed with maturing seeds. While thePPDK, AGPase, and 10 kDa prolamin genes were expressed constitutively,their expression levels in various tissues varied depending on the gene.

1. An isolated DNA comprising seed-specific promoter activity, whereinthe DNA comprises the nucleotide sequence of SEQ ID NO:
 1. 2. Theisolated DNA of claim 1, further comprising a heterologous gene operablylinked to the promoter.
 3. A vector comprising the DNA of claim
 1. 4. Avector comprising the DNA of claim
 2. 5. A plant cell transformed withthe DNA of claim
 2. 6. A plant cell transformed with the vector of claim3.
 7. A plant cell transformed with the vector of claim
 4. 8. Atransformed plant comprising the cell of claim
 6. 9. A transformed plantcomprising the cell of claim
 7. 10. A reproductive material of the plantof claim
 8. 11. A reproductive material of the plant of claim
 9. 12. Thereproductive material of claim 10, wherein the reproductive material isa seed.
 13. The reproductive material of claim 11, wherein thereproductive material is a seed.
 14. A method of expressing a gene in aseed generated from a plant cell, comprising the steps of: (a)introducing the DNA of claim 2 into the plant cell, (b) regenerating aplant from the plant cell, and (c) growing the transgenic plant tomaturity to produce seeds, wherein said gene is expressed in said seeds.15. A method of expressing a gene in a seed generated from a plant cell,comprising the steps of: (a) introducing the DNA of claim 3 into theplant cell, (b) regenerating a plant from the plant cell, and (c)growing the transgenic plant to maturity to produce seeds, wherein saidgene is expressed in said seeds.
 16. A method of expressing a gene in aseed generated from a plant cell, comprising the steps of: (a)introducing the DNA of claim 4 into the plant cell, (b) regenerating aplant from the plant cell, and (c) growing the transgenic plant tomaturity to produce seeds, wherein said gene is expressed in said seeds.17. An isolated DNA which consists of the nucleotide sequence of SEQ IDNO:8.
 18. A vector comprising the DNA of claim
 17. 19. The isolated DNAof claim 1, further comprising a 3′-untranslated region as shown in SEQID NO:
 8. 20. A recombinant DNA construct comprising the nucleotidesequence of SEQ ID NO: 1 operably linked to a heterologous gene.
 21. Arecombinant DNA construct comprising the nucleotide sequence of SEQ IDNO: 8 operably linked to a heterologous gene.